RESUMO
BACKGROUND: Thromboembolic events, including myocardial infarction (MI) or stroke, caused by the rupture or erosion of unstable atherosclerotic plaques are the leading cause of death worldwide. Although most mouse models of atherosclerosis develop lesions in the aorta and carotid arteries, they do not develop advanced coronary artery lesions. Moreover, they do not undergo spontaneous plaque rupture with MI and stroke or do so at such a low frequency that they are not viable experimental models to study late-stage thrombotic events or to identify novel therapeutic approaches for treating atherosclerotic disease. This has stymied the development of more effective therapeutic approaches for reducing these events beyond what has been achieved with aggressive lipid lowering. Here, we describe a diet-inducible mouse model that develops widespread advanced atherosclerosis in coronary, brachiocephalic, and carotid arteries with plaque rupture, MI, and stroke. METHODS: We characterized a novel mouse model with a C-terminal mutation in the scavenger receptor class B, type 1 (SR-BI), combined with Ldlr knockout (designated SR-BI∆CT/∆CT/Ldlr-/-). Mice were fed Western diet (WD) for 26 weeks and analyzed for MI and stroke. Coronary, brachiocephalic, and carotid arteries were analyzed for atherosclerotic lesions and indices of plaque stability. To validate the utility of this model, SR-BI∆CT/∆CT/Ldlr-/- mice were treated with the drug candidate AZM198, which inhibits myeloperoxidase, an enzyme produced by activated neutrophils that predicts rupture of human atherosclerotic lesions. RESULTS: SR-BI∆CT/∆CT/Ldlr-/- mice show high (>80%) mortality rates after 26 weeks of WD feeding because of major adverse cardiovascular events, including spontaneous plaque rupture with MI and stroke. Moreover, WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice displayed elevated circulating high-sensitivity cardiac troponin I and increased neutrophil extracellular trap formation within lesions compared with control mice. Treatment of WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice with AZM198 showed remarkable benefits, including >90% improvement in survival and >60% decrease in the incidence of plaque rupture, MI, and stroke, in conjunction with decreased circulating high-sensitivity cardiac troponin I and reduced neutrophil extracellular trap formation within lesions. CONCLUSIONS: WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice more closely replicate late-stage clinical events of advanced human atherosclerotic disease than previous models and can be used to identify and test potential new therapeutic agents to prevent major adverse cardiac events.
Assuntos
Modelos Animais de Doenças , Camundongos Knockout , Infarto do Miocárdio , Peroxidase , Placa Aterosclerótica , Acidente Vascular Cerebral , Animais , Infarto do Miocárdio/patologia , Infarto do Miocárdio/tratamento farmacológico , Placa Aterosclerótica/tratamento farmacológico , Camundongos , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/prevenção & controle , Peroxidase/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Receptores de LDL/genética , Receptores de LDL/deficiência , Camundongos Endogâmicos C57BL , Masculino , Ruptura Espontânea , Dieta Ocidental/efeitos adversos , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/farmacologiaRESUMO
Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.
Assuntos
Receptores Depuradores/classificação , Receptores Depuradores/fisiologia , Animais , Endocitose , Humanos , Ligantes , Camundongos , National Institute of Allergy and Infectious Diseases (U.S.)/normas , Fagocitose , Receptores Imunológicos/fisiologia , Receptores Depuradores Classe A/fisiologia , Transdução de Sinais , Terminologia como Assunto , Estados UnidosRESUMO
The HDL receptor scavenger receptor, class B type I (SR-BI) controls the structure and fate of plasma HDL. Female SR-BI KO mice are infertile, apparently because of their abnormal cholesterol-enriched HDL particles. We examined the growth and meiotic progression of SR-BI KO oocytes and found that they underwent normal germinal vesicle breakdown; however, SR-BI KO eggs, which had accumulated excess cholesterol in vivo, spontaneously activated, and they escaped metaphase II (MII) arrest and progressed to pronuclear, MIII, and anaphase/telophase III stages. Eggs from fertile WT mice were activated when loaded in vitro with excess cholesterol by a cholesterol/methyl-ß-cyclodextrin complex, phenocopying SR-BI KO oocytes. In vitro cholesterol loading of eggs induced reduction in maturation promoting factor and MAPK activities, elevation of intracellular calcium, extrusion of a second polar body, and progression to meiotic stages beyond MII. These results suggest that the infertility of SR-BI KO females is caused, at least in part, by excess cholesterol in eggs inducing premature activation and that cholesterol can activate WT mouse eggs to escape from MII arrest. Analysis of SR-BI KO female infertility raises the possibility that abnormalities in cholesterol metabolism might underlie some cases of human female infertility of unknown etiology.
Assuntos
HDL-Colesterol/metabolismo , Colesterol/toxicidade , Infertilidade Feminina/etiologia , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Receptores Depuradores Classe B/deficiência , Animais , Sobrevivência Celular , Ácido Egtázico/farmacologia , Feminino , Sistema de Sinalização das MAP Quinases , Meiose/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Corpos Polares , Receptores Depuradores Classe B/fisiologia , Estrôncio/farmacologia , beta-Ciclodextrinas/farmacologiaRESUMO
The HDL receptor SR-BI mediates the transfer of cholesteryl esters from HDL to cells and controls HDL abundance and structure. Depending on the genetic background, loss of SR-BI causes hypercholesterolemia, anemia, reticulocytosis, splenomegaly, thrombocytopenia, female infertility, and fatal coronary heart disease (CHD). The carboxy terminus of SR-BI (505QEAKL509) must bind to the cytoplasmic adaptor PDZK1 for normal hepatic-but not steroidogenic cell-expression of SR-BI protein. To determine whether SR-BI's carboxy terminus is also required for normal protein levels in steroidogenic cells, we introduced into SR-BI's gene a 507Ala/STOP mutation that produces a truncated receptor (SR-BIΔCT). As expected, the dramatic reduction of hepatic receptor protein in SR-BIΔCT mice was similar to that in PDZK1 knockout (KO) mice. Unlike SR-BI KO females, SR-BIΔCT females were fertile. The severity of SR-BIΔCT mice's hypercholesterolemia was intermediate between those of SR-BI KO and PDZK1 KO mice. Substantially reduced levels of the receptor in adrenal cortical cells, ovarian cells, and testicular Leydig cells in SR-BIΔCT mice suggested that steroidogenic cells have an adaptor(s) functionally analogous to hepatic PDZK1. When SR-BIΔCT mice were crossed with apolipoprotein E KO mice (SR-BIΔCT/apoE KO), pathologies including hypercholesterolemia, macrocytic anemia, hepatic and splenic extramedullary hematopoiesis, massive splenomegaly, reticulocytosis, thrombocytopenia, and rapid-onset and fatal occlusive coronary arterial atherosclerosis and CHD (median age of death: 9 wk) were observed. These results provide new insights into the control of SR-BI in steroidogenic cells and establish SR-BIΔCT/apoE KO mice as a new animal model for the study of CHD.
Assuntos
Córtex Suprarrenal/metabolismo , Hipercolesterolemia/genética , Células Intersticiais do Testículo/metabolismo , Fígado/metabolismo , Ovário/metabolismo , Receptores Depuradores Classe B/genética , Anemia Macrocítica/genética , Animais , Apolipoproteínas E/genética , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/mortalidade , Doença das Coronárias/genética , Doença das Coronárias/mortalidade , Oclusão Coronária/genética , Oclusão Coronária/mortalidade , Feminino , Técnicas de Introdução de Genes , Hematopoese Extramedular/genética , Immunoblotting , Lipoproteínas HDL/genética , Masculino , Camundongos , Mutação , Reação em Cadeia da Polimerase , Receptores de Lipoproteínas/genética , Reticulocitose/genética , Esplenomegalia/genética , Trombocitopenia/genética , TranscriptomaRESUMO
Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a variety of ligands, including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the U.S. National Institute of Allergy and Infectious Diseases, National Institutes of Health to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of non-self or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. The discussion and nomenclature recommendations described in this report only refer to mammalian scavenger receptors. The purpose of this article is to describe the proposed mammalian nomenclature and classification developed at the workshop and to solicit additional feedback from the broader research community.
Assuntos
Receptores Depuradores/classificação , Animais , Humanos , Receptores Depuradores/imunologia , Terminologia como AssuntoRESUMO
Mice with homozygous null mutations in the HDL receptor (scavenger receptor class B, type I, or SR-BI) and apolipoprotein E (apoE) genes [SR-BI/apoE double KO (SR-BI(-/-)/apoE(-/-) or dKO) mice] spontaneously develop occlusive, atherosclerotic coronary artery disease (CAD) and die prematurely (50% mortality at 42 d of age). Using microarray mRNA expression profiling, we identified genes whose expression in the hearts of dKO mice changed substantially during disease progression [at 21 d of age (no CAD), 31 d of age (small myocardial infarctions), and 43 d of age (extensive myocardial infarctions) vs. CAD-free SR-BI(+/-)/apoE(-/-) controls]. Expression of most genes that increased >sixfold in dKO hearts at 43 d also increased after coronary artery ligation. We examined the influence and potential mechanism of action of apolipoprotein D (apoD) whose expression in dKO hearts increased 80-fold by 43 d. Analysis of ischemia/reperfusion-induced myocardial infarction in both apoD KO mice and wild-type mice with abnormally high plasma levels of apoD (adenovirus-mediated hepatic overexpression) established that apoD reduces myocardial infarction. There was a correlation of apoD's ability to protect primary cultured rat cardiomyocytes from hypoxia/reoxygenation injury with its potent ability to inhibit oxidation in a standard antioxidation assay in vitro. We conclude that dKO mice represent a useful mouse model of CAD and apoD may be part of an intrinsic cardioprotective system, possibly as a consequence of its antioxidation activity.
Assuntos
Antioxidantes/metabolismo , Apolipoproteínas D/sangue , Doença da Artéria Coronariana/sangue , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Apolipoproteínas D/genética , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Hipóxia Celular/genética , Células Cultivadas , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Knockout , Infarto do Miocárdio/sangue , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Ratos Wistar , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismoRESUMO
A new series of potent inhibitors of cellular lipid uptake from HDL particles mediated by scavenger receptor, class B, type I (SR-BI) was identified. The series was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR) that measured the transfer of the fluorescent lipid DiI from HDL particles to CHO cells overexpressing SR-BI. The series is characterized by a linear peptidomimetic scaffold with two adjacent amide groups, as well as an aryl-substituted heterocycle. Analogs of the initial hit were rapidly prepared via Ugi 4-component reaction, and select enantiopure compounds were prepared via a stepwise sequence. Structure-activity relationship (SAR) studies suggest an oxygenated arene is preferred at the western end of the molecule, as well as highly lipophilic substituents on the central and eastern nitrogens. Compound 5e, with (R)-stereochemistry at the central carbon, was designated as probe ML279. Mechanistic studies indicate that ML279 stabilizes the interaction of HDL particles with SR-BI, and its effect is reversible. It shows good potency (IC50=17 nM), is non-toxic, plasma stable, and has improved solubility over our alternative probe ML278.
Assuntos
Alanina/análogos & derivados , Antígenos CD36/antagonistas & inibidores , Furanos/química , Compostos Heterocíclicos/química , Tetrazóis/química , Alanina/síntese química , Alanina/química , Alanina/metabolismo , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células CHO , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Lipoproteínas HDL/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Tetrazóis/síntese química , Tetrazóis/metabolismoRESUMO
We report a new series of 8-membered benzo-fused lactams that inhibit cellular lipid uptake from HDL particles mediated by Scavenger Receptor, Class B, Type I (SR-BI). The series was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR), measuring the transfer of the fluorescent lipid DiI from HDL particles to CHO cells overexpressing SR-BI. The series is part of a previously reported diversity-oriented synthesis (DOS) library prepared via a build-couple-pair approach. Detailed structure-activity relationship (SAR) studies were performed with a selection of the original library, as well as additional analogs prepared via solution phase synthesis. These studies demonstrate that the orientation of the substituents on the aliphatic ring have a critical effect on activity. Additionally, a lipophilic group is required at the western end of the molecule, and a northern hydroxyl group and a southern sulfonamide substituent also proved to be optimal. Compound 2p was found to possess a superior combination of potency (av IC50=0.10µM) and solubility (79µM in PBS), and it was designated as probe ML312.
Assuntos
Antígenos CD36/antagonistas & inibidores , Lactamas/farmacologia , Metabolismo dos Lipídeos , Animais , Antígenos CD36/fisiologia , Humanos , Lactamas/química , Relação Estrutura-AtividadeRESUMO
The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2â1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.
Assuntos
Membrana Celular/metabolismo , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Domínios PDZ/fisiologia , Receptores Depuradores Classe B/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Membrana Celular/química , Membrana Celular/genética , Células Cultivadas , Medição da Troca de Deutério , Hepatócitos/química , Hepatócitos/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Ligação Proteica/fisiologia , Receptores Depuradores Classe B/química , Receptores Depuradores Classe B/genética , Deleção de Sequência , Ressonância de Plasmônio de SuperfícieRESUMO
The HDL receptor, scavenger receptor, class B, type I (SR-BI), is a homooligomeric cell surface glycoprotein that controls HDL structure and metabolism by mediating the cellular selective uptake of lipids, mainly cholesteryl esters, from HDL. The mechanism underlying SR-BI-mediated lipid transfer, which differs from classic receptor-mediated endocytosis, involves a two-step process (binding followed by lipid transport) that is poorly understood. Our previous structure/activity analysis of the small-molecule inhibitor blocker of lipid transport 1 (BLT-1), which potently (IC(50) â¼ 50 nM) blocks SR-BI-mediated lipid transport, established that the sulfur in BLT-1's thiosemicarbazone moiety was essential for activity. Here we show that BLT-1 is an irreversible inhibitor of SR-BI, raising the possibility that cysteine(s) in SR-BI interact with BLT-1. Mass spectrometric analysis of purified SR-BI showed two of its six exoplasmic cysteines have free thiol groups (Cys251 and Cys384). Converting Cys384 (but not Cys251) to serine resulted in complete BLT-1 insensitivity, establishing that the unique molecular target of BLT-1 inhibition of cellular SR-BI dependent lipid transport is SR-BI itself. The C384S substitution reduced the receptor's intrinsic lipid uptake activity by approximately 60% without dramatically altering its surface expression, homooligomerization, or HDL binding. Thus, a small-molecule screening approach identified a key residue in SR-BI involved in lipid transport, providing a powerful springboard into the analyses of the structure and mechanism of SR-BI, and highlighting the power of this approach for such analyses.
Assuntos
Receptores Depuradores Classe B/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico Ativo , Células COS , Chlorocebus aethiops , Ciclopentanos/farmacologia , Cisteína/química , Humanos , Técnicas In Vitro , Metabolismo dos Lipídeos , Lipoproteínas HDL/metabolismo , Espectrometria de Massas , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores Depuradores Classe B/antagonistas & inibidores , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Tiossemicarbazonas/farmacologiaRESUMO
BACKGROUND AND AIMS: There are a limited number of pharmacologic therapies for coronary artery disease, and few rodent models of occlusive coronary atherosclerosis and consequent myocardial infarction with which one can rapidly test new therapeutic approaches. Here, we characterize a novel, fertile, and easy-to-use HDL receptor (SR-B1)-based model of atherogenic diet-inducible, fatal coronary atherosclerosis, the SR-B1ΔCT/LDLR KO mouse. Additionally, we test intramyocardial injection of Stromal Cell-Derived Factor-1 alpha (SDF-1α), a potent angiogenic cytokine, as a possible therapy to rescue cardiac function in this mouse. METHODS: SR-B1ΔCT/LDLR KO mice were fed the Paigen diet or standard chow diet, and we determined the effects of the diets on cardiac function, histology, and survival. After two weeks of feeding either the Paigen diet (n = 24) or standard chow diet (n = 20), the mice received an intramyocardial injection of either SDF-1α or phosphate buffered saline (PBS). Cardiac function and angiogenesis were assessed two weeks later. RESULTS: When six-week-old mice were fed the Paigen diet, they began to die as early as 19 days later and 50% had died by 38 days. None of the mice maintained on the standard chow diet died by day 72. Hearts from mice on the Paigen diet showed evidence of cardiomegaly, myocardial infarction, and occlusive coronary artery disease. For the five mice that survived until day 28 that underwent an intramyocardial injection of PBS on day 15, the average ejection fraction (EF) decreased significantly from day 14 (the day before injection, 52.1 ± 4.3%) to day 28 (13 days after the injection, 30.6 ± 6.8%) (paired t-test, n = 5, p = 0.0008). Of the 11 mice fed the Paigen diet and injected with SDF-1α on day 15, 8 (72.7%) survived to day 28. The average EF for these 8 mice increased significantly from 48.2 ± 7.2% on day 14 to63.6 ± 6.9% on day 28 (Paired t-test, n = 8, p = 0.003). CONCLUSIONS: This new mouse model and treatment with the promising angiogenic cytokine SDF-1α may lead to new therapeutic approaches for ischemic heart disease.
Assuntos
Quimiocina CXCL12 , Doença da Artéria Coronariana , Modelos Animais de Doenças , Camundongos Knockout , Receptores de LDL , Receptores Depuradores Classe B , Animais , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genética , Receptores de LDL/genética , Receptores de LDL/deficiência , Receptores Depuradores Classe B/genética , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Dieta Aterogênica , Camundongos , Função Ventricular Esquerda , Miocárdio/patologia , Miocárdio/metabolismo , Dieta HiperlipídicaRESUMO
OBJECTIVE: Deep vein thrombosis (DVT) and pulmonary embolism are frequent causes of morbidity and mortality. The goal of our study was to determine whether plasma high-density lipoprotein (HDL), which inversely correlates with the risk of cardiovascular events, affects DVT. METHODS AND RESULTS: Using a murine DVT model of inferior vena cava stenosis, we demonstrated that deficiency of the HDL receptor, scavenger receptor class B type I (SR-BI), promotes venous thrombosis. As SR-BI(-/-) mice have increased plasma cholesterol levels and abnormal HDL particles, we tested SR-BI(-/-) mice with an SR-BI liver transgene that normalizes both parameters. These mice also exhibited increased susceptibility to DVT, indicating a protective role of extrahepatic SR-BI. Mice lacking the major HDL apolipoprotein apoA-I or endothelial nitric oxide synthase (eNOS) (a downstream target of endothelial SR-BI signaling) also had a prothrombotic phenotype. Intravenous infusion of human apoA-I, an HDL component and SR-BI ligend, prevented DVT in wild-type but not SR-BI(-/-) or eNOS(-/-) mice, suggesting that its effect is mediated by SR-BI and eNOS. Intravenous apoA-I infusion abolished histamine-induced platelet-endothelial interactions, which are important for DVT initiation. CONCLUSIONS: An apoA-I (HDL)-SR-BI-eNOS axis is highly protective in DVT and may provide new targets for prophylaxis and treatment of venous thrombosis.
Assuntos
Apolipoproteína A-I/farmacologia , Receptores Depuradores Classe B/fisiologia , Trombose Venosa/prevenção & controle , Animais , Coagulação Sanguínea , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/fisiologiaRESUMO
The high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI's structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys(321)-Pro(322)-Cys(323) (CPC) motif and connect Cys(280) to Cys(334). We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys(384) to HDL binding and lipid uptake. The effects of CPC mutations on activity were context-dependent. Full wild-type (WT) activity required Pro(322) and Cys(323) only when Cys(321) was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX, or XXX mutants (X ≠ WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys(323) is deleterious, perhaps because of aberrant disulfide bond formation. Pro(322) may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for normal activity. C(384)X (X = S, T, L, Y, G, or A) mutants exhibited altered activities that varied with the side chain's size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (enhanced binding, weakened uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C(384)X mutants were BLT-1-resistant, supporting the proposal that Cys(384)'s thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories.
Assuntos
Antígenos CD36/química , Cisteína/química , Dissulfetos/metabolismo , Lipoproteínas HDL/química , Receptores de Lipoproteínas/química , Motivos de Aminoácidos , Animais , Antígenos CD36/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Ciclopentanos/farmacologia , Cisteína/metabolismo , Humanos , Lipoproteínas HDL/metabolismo , Estrutura Terciária de Proteína , Espectrometria de Massas em Tandem , Tiossemicarbazonas/farmacologiaRESUMO
Scavenger receptor class B, type I (SR-BI), a CD36 superfamily member, is an oligomeric high density lipoprotein (HDL) receptor that mediates negatively cooperative HDL binding and selective lipid uptake. We identified in the N-terminal transmembrane (N-TM) domain of SR-BI a conserved glycine dimerization motif, G(15)X(2)G(18)X(3)AX(2)G(25), of which the submotif G(18)X(3)AX(2)G(25) significantly contributes to homodimerization and lipid uptake activity. SR-BI variants were generated by mutations (single or multiple Gly â Leu substitutions) or by replacing the N-TM domain with those from other CD36 superfamily members containing (croquemort) or lacking (lysosomal integral membrane protein (LIMP) II) this glycine motif (chimeras). None of the SR-BI variants exhibited altered surface expression (based on antibody binding) or HDL binding. However, the G15L/G18L/G25L triple mutant exhibited reductions in cell surface homo-oligomerization (>10-fold) and the rate of selective lipid uptake (â¼ 2-fold). Gly(18) and Gly(25) were necessary for normal lipid uptake activity of SR-BI and the SR-BI/croquemort chimera. The lipid uptake activity of the glycine motif-deficient SR-BI/LIMP II chimera was low but could be increased by introducing glycines at positions 18 and 25. The rate of lipid uptake mediated by SR-BI/LIMP II chimeras was proportional to the extent of receptor oligomerization. Thus, the glycine dimerization motif G(18)X(3)AX(2)G(25) in the N-TM domain of SR-BI contributes substantially to the homo-oligomerization and lipid transport activity of SR-BI but does not influence the negative cooperativity of HDL binding. Oligomerization-independent binding cooperativity suggests that classic allostery is not involved and that the negative cooperativity is probably the consequence of a "lattice effect" (interligand steric interference accompanying binding to adjacent receptors).
Assuntos
Lipoproteínas HDL/metabolismo , Multimerização Proteica/fisiologia , Receptores Depuradores Classe B/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Transporte Biológico/fisiologia , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Humanos , Lipoproteínas HDL/genética , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Camundongos , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptores Depuradores Classe B/genéticaRESUMO
The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1-PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI ("target peptide") binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr(20) â Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the target peptide (K(d) = 37.0 µm), albeit with â¼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr(253) â Ala substitution. Comparison of the 1.5-Å resolution crystal structure of PDZ3 with its bound target peptide ((505)QEAKL(509)) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr(20) â Ala (PDZ1) + Tyr(253) â Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr(253) â Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr(20) â Ala (PDZ1) + Tyr(253) â Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Receptores Depuradores Classe B/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Técnicas de Introdução de Genes , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Receptores Depuradores Classe B/química , Receptores Depuradores Classe B/genética , TransgenesRESUMO
Scavenger receptor class B, type I (SR-BI), is a high-density lipoprotein (HDL) receptor, which also binds low-density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a coreceptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high-affinity binding sites (one site model). We have reinvestigated the ligand concentration dependence of (125)I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [(3)H]CE from [(3)H]CE-HDL using an expanded range of ligand concentrations (<1 µg of protein/mL, lower than previously reported). Scatchard and nonlinear least-squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects ("lattice model"). Similar results were observed for LDL. Application of the "infinite dilution" dissociation rate method established that the binding of (125)I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport, and cell signaling.
Assuntos
Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/metabolismo , Receptores Depuradores Classe B/química , Receptores Depuradores Classe B/metabolismo , Animais , Sítios de Ligação , Células CHO , LDL-Colesterol/metabolismo , Cricetinae , Cricetulus , Humanos , LigantesRESUMO
The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 µM, respectively, similar to 2.6 µM measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Receptores Depuradores Classe B/metabolismo , Substituição de Aminoácidos , Animais , Colesterol/genética , Colesterol/metabolismo , Cristalografia por Raios X , Feminino , Regulação da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Receptores Depuradores Classe B/genéticaRESUMO
Cerebrovascular and cardiovascular diseases are a major cause of morbidity and mortality. Soluble P-selectin (sP-selectin) is a biomarker for platelet/endothelial activation and is considered a risk factor for vascular disease. sP-selectin enhances procoagulant activity by inducing leukocyte-derived microparticle production and promotes activation of leukocyte integrins. However, it is not known whether it directly contributes to vascular complications. We investigated the effect of increased levels of sP-selectin on blood-brain barrier (BBB) function, stroke outcome, and atherosclerosis by comparing wild-type mice with P-sel(DeltaCT/DeltaCT) mice in which the endogenous P-selectin gene was replaced with a mutant that produces abnormally high plasma levels of sP-selectin. P-sel(DeltaCT/DeltaCT) mice presented several abnormalities, including (1) higher BBB permeability, with 25% of the animals showing differential permeability between the right and left hemispheres; (2) altered social behavior with increased aggression; (3) larger infarcts in the middle cerebral artery occlusion ischemic stroke model; and (4) increased susceptibility to atherosclerotic, macrophage-rich lesion development in both male and female mice on the apoE(-/-) genetic background. Thus, elevated sP-selectin is not only a biomarker for vascular disease, but also may contribute directly to atherosclerosis and cerebrovascular complications.
Assuntos
Aterosclerose/sangue , Barreira Hematoencefálica/metabolismo , Infarto Cerebral/sangue , Selectina-P/sangue , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/patologia , Infarto Cerebral/patologia , Feminino , Masculino , Camundongos , Selectina-P/genética , SolubilidadeRESUMO
The conserved oligomeric Golgi (COG) complex is essential for establishing and/or maintaining the structure and function of the Golgi apparatus. The Golgi apparatus, in turn, has a central role in protein sorting and glycosylation within the eukaryotic secretory pathway. As a consequence, COG mutations can give rise to human genetic diseases known as congenital disorders of glycosylation. We review recent results from studies of yeast, worm, fly and mammalian COG that provide evidence that COG might function in retrograde vesicular trafficking within the Golgi apparatus. This hypothesis explains the impact of COG mutations by postulating that they impair the retrograde flow of resident Golgi proteins needed to maintain normal Golgi structure and function.
Assuntos
Proteínas de Transporte/fisiologia , Complexo de Golgi/fisiologia , Modelos Biológicos , Proteínas/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Animais , Transporte Biológico/fisiologia , HumanosRESUMO
RATIONALE: Heart failure is a deadly and devastating disease that places immense costs on an aging society. To develop therapies aimed at rescuing the failing heart, it is important to understand the molecular mechanisms underlying cardiomyocyte structure and function. OBJECTIVE: microRNAs are important regulators of gene expression, and we sought to define the global contributions made by microRNAs toward maintaining cardiomyocyte integrity. METHODS AND RESULTS: First, we performed deep sequencing analysis to catalog the miRNA population in the adult heart. Second, we genetically deleted, in cardiac myocytes, an essential component of the machinery that is required to generate miRNAs. Deep sequencing of miRNAs from the heart revealed the enrichment of a small number of microRNAs with one, miR-1, accounting for 40% of all microRNAs. Cardiomyocyte-specific deletion of dgcr8, a gene required for microRNA biogenesis, revealed a fully penetrant phenotype that begins with left ventricular malfunction progressing to a dilated cardiomyopathy and premature lethality. CONCLUSIONS: These observations reveal a critical role for microRNAs in maintaining cardiac function in mature cardiomyocytes and raise the possibility that only a handful of microRNAs may ultimately be responsible for the dramatic cardiac phenotype seen in the absence of dgcr8.